CN113604509B - Saccharomyces bifidus fermentation product and preparation method and application thereof - Google Patents
Saccharomyces bifidus fermentation product and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9728—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a fermentation product of saccharomyces cerevisiae, a preparation method and application thereof, wherein the preparation method comprises the following steps: inoculating bifidobacterium longum into a seed culture medium, and performing anaerobic culture and activation to obtain seed liquid; inoculating the seed liquid into a fermentation culture medium, regulating the initial pH value of the fermentation liquid to 6.5-7.0, and carrying out anaerobic fermentation culture; centrifuging the obtained fermentation liquor, and taking supernatant as a fermentation product of the saccharomyces cerevisiae; the seed culture medium and the fermentation culture medium are prepared by taking 5-10 g of bean dregs and 1-5 g of bagasse, and supplementing water to 1kg. The invention adopts the bean dregs and the bagasse as the bifidobacterium culture medium, and utilizes the rich and various nutritional ingredients of the bean dregs and the bagasse to further strengthen and promote the beneficial metabolites of the bifidobacterium. The filtrate of the fermentation product of the saccharomyces cerevisiae has better application effect in cosmetics: has excellent efficacy performance of promoting the expression of genes related to barrier repair and promoting the synthesis of collagen.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a fermentation product of saccharomyces cerevisiae, a preparation method and application thereof.
Background
The fermentation product of Saccharomyces cerevisiae (BIFIDA FERMENT FILTRATE) is a metabolite obtained by fermenting Bacillus bifidus, and contains organic acids (lactic acid, citric acid, succinic acid, etc.), vitamins (B1, B2, B6, B12, pantothenic acid, folic acid, etc.), bacteriocins (mostly water-soluble polypeptides), polysaccharides, proteins, amino acids, etc.
Lactic acid in the fermentation product of the saccharomyces cerevisiae is a natural moisturizing factor, exists in the skin of a human body, and has the effects of effectively removing fine lines and wrinkles, accelerating cutin renewal and the like; folic acid can activate skin epidermis cells, promote the maintenance of moisture and the absorption of nutrient components, and reduce the keratinization speed of skin, thereby achieving the purposes of maintaining beauty and nourishing skin and tendering skin; in addition, the active ingredients such as amino acid, lipid, polysaccharide, adenosine, vitamins, trace minerals, vitamin B groups and the like can moisten and nourish skin, help skin resist the damage to the skin caused by external environment and pressure, and can promote to express multiple protein genes related to epidermis differentiation and skin barrier function, thereby achieving the effects of removing wrinkles, repairing, moisturizing and enhancing skin elasticity. Therefore, the fermentation product of the saccharomyces cerevisiae has great market application prospect. However, the preparation process of the fermentation product filtrate of the two-split yeast used for the cosmetic raw material is complex, and the fermentation growth of bifidobacteria is often promoted by continuously supplementing exogenous nutrient components, so that some exogenous additive components remain in the metabolic product, and the production cost of fermentation is increased.
Disclosure of Invention
The invention aims to provide a preparation method of a fermentation product of saccharomyces cerevisiae, which effectively improves the anti-aging capability of the obtained fermentation product by adopting bean dregs and bagasse as culture mediums, and is particularly embodied in promoting the expression of genes related to barrier repair and improving the activity of fibroblast and the expression quantity of COL-I protein. These effects are not achieved by prior art methods.
The aim of the invention is achieved by the following technical scheme:
the preparation method of the fermentation product of the saccharomyces cerevisiae comprises the following steps:
(1) Inoculating bifidobacterium longum into a seed culture medium, and performing anaerobic culture and activation to obtain seed liquid;
(2) Inoculating the seed liquid into a fermentation culture medium, regulating the initial pH value of the fermentation liquid to 6.5-7.0, and carrying out anaerobic fermentation culture;
(3) Centrifuging the fermentation liquor obtained in the step (2), and taking supernatant as a fermentation product of the saccharomyces cerevisiae;
preferably, the obtained fermentation product of the saccharomyces cerevisiae is subjected to low-temperature pasteurization, filtration and preservative addition to obtain the fermentation product of the saccharomyces cerevisiae which can be industrially used.
Bifidobacterium longum, preferably bifidobacterium longum ATCC 15697, as described in step (1);
the seed culture medium and the fermentation culture medium are prepared by taking 5-10 g of bean dregs and 1-5 g of bagasse, and supplementing water to 1kg; wherein, the bean dregs and the bagasse are crushed and then pass through a screen mesh of 40 to 80 meshes; the bean dregs contain a large amount of various nutrient elements such as protein, dietary fiber, amino acid, vitamins, minerals and the like, and provide a natural N source for the growth of bifidobacteria; in addition, the rich polysaccharide component in bagasse is utilized to provide a natural C source for the growth of bifidobacteria.
Preferably, in the step (1), the inoculation amount of the bifidobacterium longum accounts for 1-3% of the mass of the seed culture medium; the anaerobic culture temperature is 35-40 ℃ (particularly preferably 36-38 ℃), and the culture time is 20-30 hours; the concentration of Bifidobacterium longum in the obtained seed solution is 1×10 6 ～1×10 8 CFU/mL。
Preferably, in the step (2), the seed liquid accounts for 3-5% of the volume of the fermentation medium; the anaerobic fermentation culture temperature is 36-38 ℃ and the culture time is 40-60 h; adjusting the pH value of the fermentation liquor refers to adjusting by using a citric acid-sodium citrate buffer solution;
in the step (3), the centrifugation is carried out for 15-30 min at 3000-6000 r/min;
the low-temperature pasteurization is carried out, the inactivation treatment temperature is 60-80 ℃, the inactivation treatment time is 20-50 min, and the inactivation treatment of microorganisms is completed;
and filtering, namely filtering by using a ceramic membrane filter, and sequentially passing filtrate through ceramic membrane filter columns with the diameters of 1.5 mu m and 0.6 mu m to remove fine impurities.
The preservative is one or more of polyalcohol, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone, ammonium benzoate or potassium sorbate;
the polyalcohol is more than one of butanediol, pentanediol or hexanediol.
The fermentation product of the Saccharomyces cerevisiae prepared by the method can be applied to cosmetics.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention adopts the bean dregs and the bagasse as the bifidobacterium culture medium, and utilizes the rich and various nutritional ingredients of the bean dregs and the bagasse to further strengthen and promote the beneficial metabolites of the bifidobacterium.
(2) After the fermentation product is centrifugally treated, the lower layer bifidobacterium longum, the culture medium residue and the upper layer bifidobacterium longum metabolite are respectively collected; the upper layer bifidobacterium longum metabolic products are treated to obtain a fermentation product filtrate of the saccharomyces cerevisiae, and the lower layer bifidobacterium longum and the culture medium residues can be recycled, so that the product cost is further reduced.
(3) The filtrate of the fermentation product of the saccharomyces cerevisiae has better application effect in cosmetics: has excellent efficacy performance of promoting the expression of genes related to barrier repair and promoting the synthesis of collagen.
Drawings
FIG. 1 is a histogram of fold amplification of genes involved in barrier repair.
FIG. 2 is a histogram of fibroblast activity.
FIG. 3 is a histogram showing the expression level of COL-I protein.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
A preparation method of a fermentation product of a saccharomyces cerevisiae comprises the following steps:
(1) Inoculating bifidobacterium longum (ATCC 15697) into a seed culture medium for anaerobic fermentation culture to obtain seed liquid; wherein the inoculating amount of the bifidobacterium is 3% of the volume of the seed culture medium, the culture temperature is 38 ℃, the time is 30h, and the concentration of the seed liquid is 1 multiplied by 10 8 CFU/mL; the seed culture solution is prepared by taking 10g of bean dregs and 3g of bagasse, and supplementing water to 1kg; wherein, the bean dregs and bagasse are crushed and then pass through a 60-mesh screen.
(2) Inoculating the cultured seed liquid into a sterilized fermentation medium according to 5% of the volume of the medium, and regulating the pH value of the feed liquid in a fermentation tank to 7.0 by using a 0.01mol/L citric acid-sodium citrate buffer solution, wherein the anaerobic fermentation culture is performed at the temperature of 38 ℃ for 60 hours; the fermentation medium is prepared by taking 10g of bean dregs and 3g of bagasse, and supplementing water to 1kg.
(3) Respectively collecting the lower layer bifidobacterium longum, the culture medium residue and the upper layer bifidobacterium longum metabolite by utilizing the rotation speed of a horizontal centrifuge of 6000r/min and the centrifugation time of 15 min; pasteurizing the upper product, wherein the inactivation temperature is 68 ℃, and the inactivation treatment time is 30min; filtering by using a ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 mu m and 0.6 mu m to remove fine impurities; the added preservative is 0.96 percent (based on the volume of the fermentation liquid obtained in the step (2) as a calculation basis; the same shall apply below), butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone are compounded to obtain a fermentation product of the saccharomyces cerevisiae.
Example 2
A preparation method of a fermentation product of a saccharomyces cerevisiae comprises the following steps:
(1) Inoculating bifidobacterium longum (ATCC 15697) into a seed culture medium for anaerobic fermentation culture to obtain seed liquid; wherein the inoculation amount of the bifidobacterium is 2% of the volume of the seed culture medium, the culture temperature is 37 ℃, the time is 24 hours, and the concentration of the strain is 3.0x10 7 CFU/mL; the seed culture solution is prepared by taking 5g of bean dregs and 1g of bagasse, and supplementing water to 1kg; wherein, the bean dregs and bagasse are crushed and then pass through a 60-mesh screen.
(2) Inoculating the cultured seed liquid into a sterilized fermentation medium according to the volume of 4% of the culture medium, and regulating the pH value of the feed liquid in a fermentation tank to 6.6 by using a 0.01mol/L citric acid-sodium citrate buffer solution, wherein the anaerobic fermentation culture is performed for 48 hours at the temperature of 37 ℃; the fermentation medium is prepared by taking 7g of bean dregs and 2g of bagasse, and supplementing water to 1kg.
(3) The rotation speed of a horizontal centrifuge is 5000r/min, the centrifugation time is 20min, and the lower layer bifidobacterium longum, the culture medium residue and the upper layer bifidobacterium longum metabolite are respectively collected; pasteurizing the upper product, wherein the inactivation temperature is 65 ℃, and the inactivation treatment time is 30min; filtering by using a ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 mu m and 0.6 mu m to remove fine impurities; the added preservative is compounded with 0.96 percent of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone to obtain a fermentation product of the saccharomyces cerevisiae.
Example 3
A preparation method of a fermentation product of a saccharomyces cerevisiae comprises the following steps:
(1) Inoculating bifidobacterium longum (ATCC 15697) into a seed culture medium for anaerobic fermentation culture to obtain seed liquid; wherein the inoculation amount of the bifidobacterium is 1% of the volume of the seed culture medium, the culture temperature is 36 ℃, the time is 20 hours, and the concentration of the strain is 1.0x10 6 CFU/mL; the seed culture solution is prepared by taking 5g of bean dregs and 1g of bagasse, and supplementing water to 1kg; wherein, the bean dregs and bagasse are crushed and then pass through a 60-mesh screen.
(2) Inoculating the cultured seed liquid into a sterilized fermentation medium according to 2% of the mass of the medium, and regulating the pH value of the feed liquid in a fermentation tank to 6.5 by using a 0.01mol/L citric acid-sodium citrate buffer solution, wherein the anaerobic fermentation culture is performed at 36 ℃ for 40 hours; the fermentation medium is prepared by taking 5g of bean dregs and 1g of bagasse, and supplementing water to 1kg.
(3) The rotation speed of the horizontal centrifuge is 4000r/min, the centrifugation time is 25min, and the lower bifidobacterium longum, the culture medium residue and the upper bifidobacterium longum metabolite are respectively collected; pasteurizing the upper product, wherein the inactivation temperature is 65 ℃, and the inactivation treatment time is 30min; filtering by using a ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 mu m and 0.6 mu m to remove fine impurities; the added preservative is compounded with 0.96 percent of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone to obtain a fermentation product of the saccharomyces cerevisiae.
Comparative example 1
A preparation method of a fermentation product of a saccharomyces cerevisiae comprises the following steps:
(1) Inoculating bifidobacterium longum (ATCC 15697) into a seed culture medium for anaerobic fermentation culture to obtain seed liquid; wherein the inoculation amount of the bifidobacterium is 3% of the volume of a seed culture medium, the culture temperature is 38 ℃ and the time is 24 hours, and the seed culture medium is prepared by supplementing water to 1kg from 5g of skimmed milk powder, 1.5g of glucose and 1.5g of isolactose;
(2) Inoculating the cultured seed liquid into a sterilized fermentation medium for anaerobic fermentation culture, and regulating the pH value of the feed liquid in a fermentation tank to 6.5 by using a 0.01mol/L citric acid-sodium citrate buffer solution, wherein the anaerobic fermentation culture is carried out at the temperature of 38 ℃ for 48 hours; the fermentation culture medium comprises 3g of glucose, 1.5g of casein peptone, 3.0g of beef peptone, 2.0g of hydrolyzed milk protein and 1kg of water;
(3) Using a horizontal centrifuge with the rotating speed of 4000r/min and the centrifugation time of 25min, taking the upper filtrate to carry out pasteurization, wherein the inactivation temperature is 65 ℃, and the inactivation treatment time is 30min; filtering by using a ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 mu m and 0.6 mu m to remove fine impurities; the added preservative is compounded with 0.96 percent of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone to obtain a fermentation product of the saccharomyces cerevisiae.
Efficacy testing
The results of the fermentation products obtained in examples 1 to 3 and comparative example 1 were examined and compared for their effects in promoting the expression of genes related to barrier repair, promoting collagen synthesis, etc.:
1. promoting barrier repair-associated gene expression
Barrier repair-associated genes (TGM 1, FLG, LOR, IVL and ABCA 12) expression were determined using human keratinocytes (HaCat cells) as a model.
Transglutaminase 1 gene TGM1: the coded membrane-associated calcium-dependent thiolase can resist the hydrolysis of protease, is a key step of the terminal differentiation of keratinocytes to form a keratinocyte envelope, and is the material basis of skin barrier function.
Silk polymeric protein FLG: is the key of skin barrier, can reduce the invasion of antigen and TEWL, can release acidic substances to reduce the pH value of the skin, and can inhibit the growth of bacteria.
Paphiopedilum LOR: is the main component of the epidermal granule cell keratinocyte transparent protein granule, and FLG gene mutation affects the terminal differentiation of keratinocyte and abnormal expression of the keratinocyte to cause the skin barrier function to be damaged.
Endo-coat protein IVL: cross-linking and deposition inside cell membrane under the catalysis of transglutaminase TGK forms a water insoluble keratinized envelope.
ABCA12 gene: as an ABC family liposome transporter, located at lamellar body limiting membranes, participate in lamellar body formation, playing an important role in maintaining normal skin barrier function.
The test steps are as follows:
(1) Cell inoculation: according to 2.5X10 5 Cell/well density was seeded into 6-well plates in an incubator (37 ℃,5% co) 2 95% rh) was cultured overnight.
(2) Preparing liquid: the fermentation products of the examples and comparative examples were prepared into a 5% volume fraction solution using complete medium (mem+10% fbs) as a solvent, and used as a working solution for a test substance for fluorescent quantitative PCR test.
(3) Administration: 3 repeated holes are arranged in each group, when the plating rate of cells in the 6-hole plate reaches 40% -50%, grouping drug delivery is carried out, and the cells are placed in an incubator (37 ℃ C., 5% CO) 2 95% RH) for 24h.
(4) Collecting cells: after incubation of the samples for 24h, PBS wash was performed, 1 mL/well, and washed twice. After washing, 1mL RNAiso Plus was added to each well, and after cell lysis by blowing, the cells were collected in 1.5mL enzyme-free EP tubes and stored in a-80℃refrigerator.
(5) Fluorescent quantitative PCR test: RNA extraction operation is carried out according to RNAiso Plus instruction, reverse transcription operation is carried out according to a reverse transcription kit, and fluorescent quantitative PCR reaction system preparation and on-machine detection are carried out according to SYBR Premix Ex TaqTM II fluorescent dye (TaKaRa DRR 081A) product instruction.
(6) And (3) calculating results: by 2 -△△CT The method performs result calculation.
The results of the fluorescence quantitative PCR detection of the barrier-associated genes by the fermentation products of examples 1-3 and comparative example 1 are shown in FIG. 1, and according to the results shown in FIG. 1, the expression of barrier repair-associated genes TGM1, FLG, LOR, IVL and ABCA12 is significantly promoted in examples 1-3 at a concentration of 5%, which is higher than that of comparative example 1.
2. Collagen synthesis
When the skin is irradiated by ultraviolet rays, excessive active oxygen is generated in cells, so that the expression of senescence-associated genes is caused, inflammatory cascade reaction is induced, the expression amount of elastin and collagen is reduced, and the skin is aged, such as loose skin, wrinkles and the like. UVA stimulates human skin fibroblasts, resulting in degradation of collagen, particularly type I collagen (COL-I).
The test was intended to evaluate whether the fermentation products of examples 1 to 3 and comparative example 1 have an accelerating effect on the secretion of COL-I by measuring the content of COL-I in fibroblasts after UVA stimulation, and comparing the content change with the solvent control group.
The specific test steps of the fibroblast toxicity test are as follows:
(1) Cell culture: preparing cell suspension 24 hr before test, inoculating the cell suspension into 96-well cell culture plate with 100 μl/well, 8000 cells/well, 37deg.C, 5% CO 2 Culturing for 24h.
(2) Exposure: the crude culture solution was discarded from the wells, 100. Mu.L of test samples of different concentrations were added to each well (test samples were prepared by adding an appropriate amount of complete medium (DMEM+10% FBS) to the fermentation product of example 1, and 0.5%, 1.0%, 5.0%, 10%, 15%, 20%, 30%, 40% by volume of test sample solution) to a blank and 100. Mu.L of complete medium (DMEM+10% FBS) was added. 37 ℃,5% CO 2 Incubators were incubated for 24h. Cell morphology and properties were observed under a phase contrast inverted microscope.
(3) MTT test: mu.L of 5mg/mL MTT solution was added to each well, and the incubator was incubated for 3.+ -. 0.5h. Removing liquid in the wells, adding 100 mu L of DMSO into each well, placing the wells in an oscillator for oscillation for 10-15 min, and measuring the absorption value at 570nm wavelength of an enzyme-labeled instrument.
(4) And (3) data processing: the data of each group are expressed as mean.+ -. Standard deviation, and the relative cell activity (Viability) of each group is calculated based on the cell activity of the control group as 100%.
The cells used in this test were human skin fibroblasts (HFF-1) and were purchased from Shanghai cell bank, national academy of sciences of life sciences. Example 1 at various concentrations the relative cellular activity of human skin fibroblasts in the samples is shown in figure 2, and compared to the blank sample set, the relative cellular activity of human skin fibroblasts at 20% of example 1 is 73.98% (industry considers relative cellular activity above 70% to be a safe concentration). Therefore, the addition amount of the composition of the invention is 1 to 20 percent of the safe administration concentration of cells.
3. Detection of COL-I content
(1) Inoculating: human skin fibroblasts were cultured at 5.0X10 4 Is inoculated into 6-well plates at 37 ℃ with 5% CO 2 Incubating the incubator overnight;
(2) Administration: when the cell plating rate in the 6-hole plate reaches 50% -60%, four groups (a negative control group, a blank control group, a tested sample group 1 and a tested sample group 2) are administered, and 3 compound holes are arranged in each group. Complete medium (DMEM+10% FBS) was added to each of the negative control group and the blank control group, and complete medium (DMEM+10% FBS) containing 5% (v/v) and 10% (v/v) concentration of the test substance was added to each of the test sample groups 1 and 2. 37 ℃,5% CO 2 The incubator continues to incubate for 24 hours.
(3) And (3) irradiation: 40 mJ.cm for sample group 1, sample group 2, and negative control group -2 UVA irradiation of (C), ultraviolet irradiation dose (mJ.cm) -2 ) =intensity of radiation (mW/cm 2 ) Irradiation time (min), blank group was not irradiated. After irradiation, each group of media was replaced with complete media (dmem+10% fbs), 37 ℃,5% co 2 The incubator cultures for 24 hours.
(4) COL-I content detection: the supernatant was collected and stored at-80℃and the cytokine was assayed using ELISA kit.
(5) Data analysis: data are expressed as mean ± standard deviation, data are analyzed using SPSS, if p <0.05 is statistically significant considering differences.
The results of the experiment are shown in FIG. 3, and the products of examples 1-3 and comparative example 1 of the present invention have a significant effect of increasing the expression level of COL-I protein (p < 0.05) at 10% of the administration dose, compared with the negative control (UVA+) (UVA irradiation).
According to the test of promoting the expression of barrier repair genes and promoting the formation of collagen, the filtrate of the fermentation product of the saccharomyces cerevisiae has the anti-aging effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. The preparation method of the fermentation product of the saccharomyces cerevisiae is characterized by comprising the following steps:
(1) Inoculating bifidobacterium longum into a seed culture medium, and performing anaerobic culture and activation to obtain seed liquid;
(2) Inoculating the seed liquid into a fermentation culture medium, regulating the initial pH value of the fermentation liquid to 6.5-7.0, and carrying out anaerobic fermentation culture;
(3) Centrifuging the fermentation liquor obtained in the step (2), and taking supernatant as a fermentation product of the saccharomyces cerevisiae;
the seed culture medium and the fermentation culture medium are prepared by taking 5-10 g of bean dregs and 1-5 g of bagasse, and supplementing water to 1kg.
2. The method of manufacturing according to claim 1, characterized in that: the obtained fermentation product of the saccharomyces cerevisiae is subjected to low-temperature pasteurization, filtration and preservative addition to obtain the fermentation product of the saccharomyces cerevisiae which can be industrially used.
3. The method of manufacturing according to claim 1, characterized in that: the bifidobacterium longum in the step (1) is bifidobacterium longum ATCC 15697.
4. The method of manufacturing according to claim 1, characterized in that: the bean dregs and bagasse are crushed and then pass through a screen mesh of 40-80 meshes.
5. The method of manufacturing according to claim 1, characterized in that: in the step (1), the inoculation amount of the bifidobacterium longum accounts for 1-3% of the mass of the seed culture medium; the anaerobic culture temperature is 35-40 ℃ and the culture time is 20-30 h; the concentration of Bifidobacterium longum in the obtained seed solution is 1×10 6 ～1×10 8 CFU/mL。
6. The method of manufacturing according to claim 1, characterized in that: in the step (2), the seed liquid accounts for 3-5% of the volume of the fermentation medium; the anaerobic fermentation culture temperature is 36-38 ℃ and the culture time is 40-60 h.
7. The method of manufacturing according to claim 1, characterized in that: in the step (3), the centrifugation is carried out for 15-30 min at 3000-6000 r/min.
8. The preparation method according to claim 2, characterized in that:
the low-temperature pasteurization is carried out, the inactivation treatment temperature is 60-80 ℃, and the inactivation treatment time is 20-50 min;
the filtering is carried out by utilizing a ceramic membrane filter to filter, and filtrate sequentially passes through ceramic membrane filter columns with the diameters of 1.5 mu m and 0.6 mu m;
the preservative is one or more of polyalcohol, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone, ammonium benzoate or potassium sorbate;
the polyalcohol is more than one of butanediol, pentanediol or hexanediol.
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